Restriction enzymes, particularly the Type II restriction endonucleases, are used to cut DNA sequences at specific locations. The enzymes carry this out by catalyzing the hydrolysis of phosphodiester linkages (the "scissile linkages") in each polynucleotide strand of a DNA molecule. The Type II restriction endonucleases are valuable for both the analysis of DNA molecules and for recombinant DNA procedures, and there is an ongoing effort to develop new enzymes. See, e.g., N. Brown and E. Walsby, U.S. Pat. No. 4,871,664. Restriction enzymes can be identified by recognition site (that is, the DNA sequence which is recognized and cleaved by a particular enzyme). Knowing the cleavage sites of the various restriction enzymes available enable researchers to cut DNA at the desired location through the selection of the proper enzyme.
A problem with restriction enzymes is that some recognition sites which should be cut by particular restriction enzymes are not. Resistant Eco RII restriction sites exist (S. Hattman et al., J. Virol. 32, 845 (1979)) in .0.X174 DNA that can be cleaved in the presence of an uncharacterized, heterologous "stimulator DNA". Resistant Eco RII sites have also been reported (D. H. Kreuger, et al., Nucleic Acids Res. 16, 3997 (1988); C. Pein et al, FEBS Letters 245, 141 (1989)) in phage T3 and T7 DNAs. These were cleaved in the presence of a high density of cleavable Eco RII DNA sites so the authors suggest that at least two bound recognition sites are needed for cleavage (Kruger et al., supra). No kinetics were measured, however, and the basis for the activation was not pursued. The authors reported that spermidine does not affect the Eco RII cleavage of the resistant sites. However, since the concentration range over which spermidine activates cleavage of slow and resistant sites is narrow, the effect could easily have been missed; the concentrations of spermidine used in the study (Kruger et al., supra) were not reported.
Also PaeR7, a type II restriction enzyme from Pseudomonas aeruginosa, shows a resistant site in Ad2 DNA in the presence of other susceptible Ad2 DNA sites (T. Gingeras and J. Brooks, Proc. Natl. Acad. Sci. USA 80, 402 (1983)); resistance was overcome by replacement of upstream sequences with sequence from a different source. When the gene for PaeR7 was expressed in E. coli, the bacteria were unable to restrict the growth of incoming phage even though cell extracts displayed the expected restriction activity on the phage DNA (Gingeras et al., supra); the authors speculate that this may indicate the presence of a control element.
The present invention is based on our ongoing research in the regulation of restriction enzyme activity.